The invention relates to the fields of microfluidics and cellular assays.
Better understanding of normal and pathogenic processes at the cellular level increasingly requires sensitive analysis of homogenous samples often containing only a few cells. One critical aspect of such analysis, considering the small amounts of target molecules contained in each cell, is to avoid excessive sample dilution and minimize losses. In addition, access to intracellular contents generally requires cell lysis, and current macroscopic approaches that work well on large samples cannot achieve controlled mixing of two solutions in volumes comparable to the cell volume. Cells are the basic structural and functional units of living organisms and analyzing the composition and behavior of individual cells is fundamental for understanding the physiology and pathology of any organism. Still, the majority of the biochemical methods employed in current biological research use samples containing several thousands of cells and as a result can only extract averaged information from the populations. Such global information is not always relevant to processes taking place in individual cells and concerted efforts are made to develop new techniques for quantitative analysis at single cell level.
In order to obtain comprehensive pictures of cell function, simultaneous examination of the expression of thousands of genes may be necessary. This examination is made possible by the development of microarray techniques for DNA and RNA. In general, simple knowledge of the gene sequence or the quantity of gene expression may be insufficient to predict biological functions or provide appropriate diagnostic information. Thus, several techniques to enable efficient and highly parallel identification, measurement, and analysis of proteins have been developed (e.g. protein-chip array).
Most array methods require concentrations of material above a certain threshold (e.g., 2 μg at 0.02 μg/μl concentration for MRNA analysis), and few methods can work with samples as small as 1000 cells. Flow cytometry can address issues of heterogeneity at the single cell level, but only for a small number of targets, which is limited by the number of fluorescent dyes available. In the case of genes, another limitation in the use of fluorescent probes comes from the requirement for a priori knowledge of the sequence of interest. Consequently, it has not yet been possible to address the issue of heterogeneity using current techniques for global analysis at the single cell level.
Previously reported techniques for single cell biochemical analysis involved the micropipette aspiration of the cell content, lysis of cells, and release of the molecules of interest in the surrounding environment form where they could be detected by capillary electrophoresis or electrochemical methods. Still, these methods cannot control the diffusion of the molecules into the surrounding medium and thus they are prone to errors when performing quantitative measurements. Some improvement may come from confining the diffusion to smaller volumes, e.g., by the use of microfluidic devices where intracellular contents from one or more cells have been released in a microchannel. In these systems, the progressively diminishing concentration of the molecule of interest due to the combined effects of diffusion and drift with time and distance from the source, complicate the attempts for quantification. Moreover, the reverse situation, where molecules from a fluid stream are slowly captured by one cell, locally decreasing their concentration, are difficult to probe and quantify under flow conditions. One way to address the sample dilution problem is by isolating cells in vials with volumes comparable to mammalian cell volume. Previously approaches used micro-vials, like the ones formed at the tip of micropipettes after etching. Problems with the control of fluid evaporation, and the lack of visual control of the cells in vials during experiments due to the geometry of the vial, limit the utility of such methods.
Thus, there is a need for new devices and methods for analyzing the contents of individual cells that have increased sensitivity.